Biomedical subjects
Publications and source records attributed to M Ghannoum.
In this study, the voriconazole susceptibilities of dermatophyte isolates obtained from a worldwide tinea capitis trial were compared to their susceptibilities to fluconazole and griseofulvin. The MIC ranges of voriconazole, fluconazole, and griseofulvin, were 0.002 to 0.06 microg/ml, 0.25 to 32 microg/ml, and 0.125 to 2.0 microg/ml, respectively.
Although standard conditions are available for testing the susceptibilities of filamentous fungi to antifungal agents by the Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards) broth microdilution assay, quality control (QC) MIC limits have not been established for any mold-agent combination. This multicenter (eight-center) study documented the reproducibility of tests for one isolate of Paecilomyces variotii ATCC MYA-3630 and 11 other mold isolates (three isolates of Aspergillus fumigatus; two isolates of A. terreus; one isolate each of A. flavus, A. nidulans, Fusarium moniliforme, and F. solani; and two isolates of Scedosporium apiospermum) by the CLSI reference broth microdilution method (M 38-A document). Control limits (amphotericin B, 1 to 4 microg/ml; itraconazole, 0.06 to 0.5 microg/ml; posaconazole, 0.03 to 0.25 microg/ml; voriconazole, 0.015 to 0.12 microg/ml) for the selected QC P. variotii ATCC MYA-3630 were established by the analysis of replicate MIC results. Reference isolates and corresponding MIC ranges were also established for 6 of the 12 molds evaluated. MIC limits were not proposed for the other five molds tested due to low testing reproducibility for these isolates.
Oral epithelial cell-derived human beta-defensins-1, -2, and -3 participate in innate immune responses against Candida. We hypothesized that these peptides utilize several mechanisms for protection. Recombinant hBD-1 and -2 were produced with the use of an insect cell/baculovirus expression system, while rhBD-3 was expressed as a fusion protein in E. coli. RhBD-2 and -3 were more effective at killing the candidal species at low micromolar concentrations than was rhBD-1, except for C. glabrata. While this species was relatively resistant to rhBD fungicidal activity, its adherence to oral epithelial cells was strain-specifically inhibited by the rhBDs. C. albicans hyphae were important in regulating hBD2 and -3 mRNA expression in primary human oral epithelial cells. Confocal microscopy of rhBD-2-challenged C. albicans suggests disruption of the fungal membrane. Results support the hypothesis that hBDs control fungal colonization through hyphal induction, direct fungicidal activity, and inhibition of candidal adherence.
BACKGROUND: Tinea capitis, a fungal infection of the scalp, is of increasing public health importance, and Trichophyton tonsurans has become the primary causative agent in North America. OBJECTIVES: To determine the prevalence of dermatophyte-positive scalp cultures among elementary schoolchildren in Cleveland, Ohio, describe predisposing factors, and measure the antifungal susceptibility of isolates collected. OBSERVATIONS: A total of 937 children from 8 Cleveland elementary schools were cultured for the presence of dermatophytes; 122 children (13%), all of whom were African American, had dermatophyte-positive cultures of the scalp. Sixty percent of cases were asymptomatic, indicating a carrier state. Race, scaling, and the use of anti-dandruff shampoo were associated with increased likelihood of infection. T tonsurans was the only organism isolated (except 1 Microsporum canis isolate). All isolates were susceptible to fluconazole, griseofulvin, itraconazole, and terbinafine. CONCLUSIONS: T tonsurans was the predominant dermatophyte isolated. Further multicenter studies are needed to confirm the predominance of dermatophyte-positive scalp cultures among African American children and to determine modifiable and preventable risk factors.
The most important role of susceptibility testing is to identify potentially resistant isolates for the agent being evaluated. Standard testing guidelines recently have been proposed for antifungal susceptibility testing of filamentous fungi (molds). This collaborative (eight centers) study evaluated further newly proposed guidelines (NCCLS, proposed standard M38-P, 1998) and other testing conditions for antifungal susceptibility testing of Aspergillus spp. to itraconazole and three new triazoles, posaconazole (SCH56592), ravuconazole (BMS-207147), and voriconazole. MICs of itraconazole, posaconazole, ravuconazole, and voriconazole for 15 selected isolates of three species of Aspergillus (A. fumigatus, A. flavus, and A. terreus) with well documented in vitro, clinical, or animal data were determined in each center by using four medium formulations (standard RPMI-1640 [RPMI], RPMI with 2% dextrose, antibiotic medium 3 [M3], and M3 with 2% dextrose) and two criteria of MIC determination (complete [MIC-0s] and prominent [MIC-2s] growth inhibition) at 24, 48, and 72 h. The highest reproducibility (92 to 99%) was seen with the standard RPMI and M3 media. Moreover, the distinction between itraconazole-resistant (MICs of >8 microg/ml for clinically resistant strains) and -susceptible (MICs of 0.03 to 1 microg/ml) isolates, as well as between a voriconazole-resistant laboratory mutant and other isolates (voriconazole MICs of 2 to >8 versus 0.12 to 2 microg/ml), was more consistently evident with the standard RPMI medium and when MIC-0s were determined at 48 h. These results provide further refinement of the testing guidelines for susceptibility testing of Aspergillus spp. and warrant consideration for inclusion in the future NCCLS document M38-A.
A comparative study of conventional amphotericin B, Abelcet and AmBisome was performed using a microdilution format of the NCCLS M27-A methodology for susceptibility testing against 300 fungal isolates (152 yeasts, 148 filamentous fungi) in both RPMI-1640 and antibiotic medium #3 (AB3). The clinical isolates included Candida albicans (n=54), Candida glabrata (n=25), Candida parapsilosis (n=23), Candida krusei (n=19), Candida lusitaniae (n=14), Cryptococcus neoformans (n=5), Candida tropicalis (n=12), Aspergillus flavus (n=34), Aspergillus fumigatus (n=46) and 68 other filamentous fungi encompassing 22 different genera. The minimal inhibitory concentrations (MIC) for all drugs were defined as the lowest concentrations in which there was no visible growth. MICs were determined after 48 h for yeasts and 72 h for filamentous fungi. The mean MICs +/- standard error (microg/ml) for yeasts and filamentous fungi, respectively, were: Abelcet, 0.51+/-0.21, 4.34+/-0.61; AmBisome, 1.28+/-0.24, 5.68+/-0.57; amphotericin B, 0.29+/-0.11, 1.12+/-0.19, respectively. Overall, against both yeasts and filamentous fungi Abelcet proved to have more potent antifungal activity than AmBisome. Using AB3 as opposed to RPMI-1640 generally produced lower MIC values but did not have any effect on the order of relative activity with all of the antifungal agents tested. In conclusion, our data shows that Abelcet is more active than AmBisome against pathogenic yeast and filamentous fungi when assayed in AB3 in vitro. Comparison of the activities of these antifungals in experimental animal models is necessary to determine whether these in vitro findings are correlated with in vivo efficacy.
Development of new approaches for treatment of invasive fungal infections encompasses new delivery systems for approved and investigational compounds, as well as exploiting the cell membrane, cell wall and virulence factors as putative antifungal targets. Novel delivery systems consisting of cyclodextrins, cochleates, nanoparticles/nanospheres and long circulating ('stealth') liposomes, substantially modulate the pharmacokinetics of existing compounds, and may also be useful to enhance the delivery of antifungal agents to sites of infection. Further insights into the structure-activity relationship of the antifungal triazoles that target the biosynthesis of ergosterol in the fungal cell membrane have led to the development of highly potent broad spectrum agents, including posaconazole, ravuconazole and voriconazole. Similarly, a novel generation of cell-wall active semisynthetic echinocandin 1,3 beta-glucan inhibitors (caspofungin, FK463, and VER-002) has entered clinical development. These agents have potent and broad-spectrum activity against Candida spp, and potentially useful activity against Aspergillus spp. and Pneumocystis carinii. The ongoing convergence of the fields of molecular pathogenesis, antifungal pharmacology and vaccine development will afford the opportunity to develop novel targets to complement the existing antifungal armamentarium.
The effects of voriconazole on the growth, morphology and lipids of Candida glabrata were studied. MIC data showed that voriconazole was up to 32- to 64-fold more active than fluconazole in its ability to inhibit various C. glabrata strains. Voriconazole inhibited the growth of C. glabrata in a dose-dependent fashion. Electron microscope examination showed that voriconazole treatment affected the external and internal morphology of C. glabrata. Treatment of C. glabrata with voriconazole inhibited ergosterol synthesis and led to accumulation of methylated sterols. In contrast, no significant difference in phospholipid composition was observed between treated and untreated cells.
Little is known of the antifungal susceptibility patterns and molecular epidemiology of Cryptococcus neoformans from tropical regions. We studied 164 clinical isolates of C. neofomans from 120 Ugandan AIDS patients with cryptococcal meningitis by analyzing their electrophoretic karyotypes and antifungal susceptibility profiles. Computer-assisted analysis of karyotype patterns was performed to generate dendrograms. MICs of fluconazole and flucytosine were determined by reference methods. A total of 43 distinguishable DNA types were identified among the 164 isolates. Only 30 patients (25%) were infected with their own unique strain of c. neoformans, whereas 75% of the patients shared their infecting strain with at least one other patient. Among 17 patients with more than one CSF isolate of C. neoformans, sequential isolates were identical or highly related in 12 (71%) and were different in five patients (29%). The isolates were susceptible to both fluconazole and flucytosine and there were no instances in which a stepwise increase in either fluconazole or flucytosine MICs was observed among serial isolates. These findings suggest that the epidemiology of cryptococcal disease in AIDS patients from tropical regions may be somewhat different from that observed in more temperate climates.
Research on fungi that cause opportunistic infections has increased dramatically during the past few years, largely because these organisms cause significant morbidity and mortality. Most of this research has focused on defining the virulence factors produced by these pathogens, as well as developing methods for the diagnosis of fungal diseases. With regard to studies on the biology of Candida albicans, it is now possible to isolate genes, disrupt their expression, and observe the specific effects of gene disruption on virulence and growth of the organism. Moreover, growth and virulence of this pathogen is also being studied and the effect of environmental factors on gene expression investigated. This subject is especially important in view of the fact that C. albicans can colonize and invade a number of sites in the human body. Thus, its ability to grown in the oral and vaginal tracts, as well as in blood, requires the organism to adapt to a variety of environmental stresses. Here we present observations on the growth, morphogenesis and virulence of the opportunistic fungi C. albicans and Aspergillus fumigatus.
Voriconazole (UK-109,496) is a novel triazole derivative with potent broad-spectrum activity against various fungi, including some that are inherently resistant to fluconazole, such as Candida krusei. In this study we compared the effect of subinhibitory concentrations of voriconazole and fluconazole on sterol biosynthesis of fluconazole-resistant and -susceptible Candida albicans strains, as well as C. krusei, in an effort to delineate the precise mode of action of voriconazole. Voriconazole MICs ranged from 0.003 to 4 microg/ml, while fluconazole MICs ranged from 0.25 to >64 microg/ml. To investigate the effects of voriconazole and fluconazole on candidal sterols, yeast cells were grown in the absence and presence of antifungals. In untreated C. albicans controls, ergosterol was the major sterol (accounting for 53.6% +/- 2.2% to 71.7% +/- 7.8% of the total) in C. albicans and C. krusei strains. There was no significant difference between the sterol compositions of the fluconazole-susceptible and -resistant C. albicans isolates. Voriconazole treatment led to a decrease in the total sterol content of both C. albicans strains tested. In contrast, exposure to fluconazole did not result in a significant reduction in the total sterol content of the three candidal strains tested (P > 0.5). Gas-liquid chromatographic analysis revealed profound changes in the sterol profiles of both C. albicans strains and of C. krusei in response to voriconazole. This antifungal agent exerted a similar effect on the sterol compositions of both fluconazole-susceptible and -resistant C. albicans strains. Interestingly, a complete inhibition of ergosterol synthesis and accumulation of its biosynthetic precursors were observed in both strains treated with voriconazole. In contrast, fluconazole partially inhibited ergosterol synthesis. Analysis of sterols obtained from a fluconazole-resistant C. albicans strain grown in the presence of different concentrations of voriconazole showed that this agent inhibits ergosterol synthesis in a dose-dependent manner. In C. krusei, voriconazole significantly inhibited ergosterol synthesis (over 75% inhibition). C. krusei cells treated with voriconazole accumulated the following biosynthetic intermediates: squalene, 4,14-dimethylzymosterol, and 24-methylenedihydrolanosterol. Accumulation of these methylated sterols is consistent with the premise that this agent functions by inhibiting fungal P-450-dependent 14alpha-demethylase. As expected, treating C. krusei with fluconazole minimally inhibited ergosterol synthesis. Importantly, our data indicate that voriconazole is more effective than fluconazole in blocking candidal sterol biosynthesis, consistent with the different antifungal potencies of these compounds.
The effects of voriconazole on the growth, ultrastructure, and leakage of cytoplasmic materials of Candida species were investigated. MIC data showed that voriconazole was more active than fluconazole. Exposure of yeast to voriconazole caused growth inhibition, cell wall thinning, and cell membrane degradation. Neither cell collapse nor release of cytoplasmic materials was observed in the treated cells.
We have developed a microdilution method for measuring the susceptibility of Cryptococcus neoformans to fluconazole. The present study evaluated the interlaboratory agreement of the results for the microdilution method obtained at three different sites and compared this method with the National Committee for Clinical Laboratory Standards M27-P reference method. Excellent interlaboratory agreement among the results obtained at the three sites was achieved with this method (83 and 96% agreement within 1 and 2 log2 dilutions, respectively). An overall agreement of 90% between the microdilution method and the M27-P method was observed, demonstrating the comparability of the two methods. However, there are inherent problems with the M27-P method in relation to measuring C. neoformans susceptibility, including suboptimal growth of the organism in RPMI 1640, a longer incubation period, and a narrow range of MICs. On the basis of these data, the microdilution method tested in this study is recommended for inclusion in the National Committee for Laboratory Standards method for testing the antifungal susceptibility of C. neoformans.
The minimum inhibitory concentration values for a group of pyrimidine derivatives were determined for Gram-positive and Gram-negative bacteria and yeast. The active compounds were further screened. The effect of these compounds on growth and morphology was tested, and their structural antimicrobial activity is discussed.
The mode of action of the antimicrobial agent, 5-bromo-5-nitro-1,3-dioxane (bronidox), was studied in detail for gram-positive and gram-negative bacteria, yeast and fungi. The studies included MIC testing, thiol inhibition of activity, intracellular leakage, oxygen consumption, incorporation of 3H-uridine, scanning electron microscopy, inhibition of enzyme activity (papain) and in vitro oxidation of thiols to disulfides. It appears that the primary mode of action of bronidox is the same as, or similar to, that of bronopol, i.e. the oxidation of essential protein thiol causing inhibition of enzyme activity and subsequent inhibition of microbial growth.
Fifty-three isolates of Candida albicans, representing seven strain types, were tested for their proteinase production and this parameter was correlated with adherence to buccal epithelial cells in 23 isolates and lethality to mice for 14 isolates. Variation in proteinase production and adherence existed both among isolates of the same strain type and different strain types. All isolates tested, irrespective of strain type or source, excreted an inducible proteinase and showed a tendency towards epithelial adherence. A correlation was found between adherence, proteinase production and pathogenicity. The C. albicans isolates which adhered most strongly to buccal epithelial cells had the highest relative proteinase activities and were most pathogenic. This was obvious with strain type--C--, which had a higher prevalence than other strain types in both patients and control groups. These results emphasize the role played by some specific properties of certain strains of C. albicans in the pathogenesis of candidosis.
Candida albicans grown in dexamethasone (DXM) shows an apparent increase in dry weight. This increase, however, represents an artefact due to entrapment and incorporation of DXM by the yeast. Thus opportunistic infections by C. albicans which are promoted by DXM must be due entirely to effects other than growth enhancement of the organism.